Effects of Very Low Dose Fast Neutrons on Cell Membrane And Secondary Protein Structure in Rat Erythrocytes

<div><p>The effects of ionizing radiation on biological cells have been reported in several literatures. Most of them were mainly concerned with doses greater than 0.01 Gy and were also concerned with gamma rays. On the other hand, the studies on very low dose fast neutrons (VLDFN) are rare. In this study, we have investigated the effects of VLDFN on cell membrane and protein secondary structure of rat erythrocytes. Twelve female <i>Wistar</i> rats were irradiated with neutrons of total dose 0.009 Gy (²⁴¹Am-Be, 0.2 mGy/h) and twelve others were used as control. Blood samples were taken at the 0, 4th, 8th, and 12th days postirradiation. Fourier transform infrared (FTIR) spectra of rat erythrocytes were recorded. Second derivative and curve fitting were used to analysis FTIR spectra. Hierarchical cluster analysis (HCA) was used to classify group spectra. The second derivative and curve fitting of FTIR spectra revealed that the most significant alterations in the cell membrane and protein secondary structure upon neutron irradiation were detected after 4 days postirradiation. The increase in membrane polarity, phospholipids chain length, packing, and unsaturation were noticed from the corresponding measured FTIR area ratios. This may be due to the membrane lipid peroxidation. The observed band shift in the CH₂ stretching bands toward the lower frequencies may be associated with the decrease in membrane fluidity. The curve fitting of the amide I revealed an increase in the percentage area of α-helix opposing a decrease in the β-structure protein secondary structure, which may be attributed to protein denaturation. The results provide detailed insights into the VLDFN effects on erythrocytes. VLDFN can cause an oxidative stress to the irradiated erythrocytes, which appears clearly after 4 days postirradiation.</p></div

Proposed Band assignments of the FTIR spectrum of rat erythrocytes on the 4000–400 cm⁻¹ spectral range.

<p>Note. ν = bond stretch; s = symmetric vibration; as = asymmetric vibration; δ = bending vibration.</p><p>Proposed Band assignments of the FTIR spectrum of rat erythrocytes on the 4000–400 cm⁻¹ spectral range.</p

Integrated areas and the percent changes of O-H and N-H stretching bands (3500–3040 cm⁻¹) of the rat erythrocytes FTIR spectra of control and irradiated groups at 0, 4, 8, and 12 days postirradiation.

<p>Parameters (mean ± SD)×10⁻³ were obtained by integrating the bands of second derivative of FTIR spectra,</p><p>^asignificant change to the control values (p < 0.05),</p><p>^bΔchange (%) are the percent change of irradiated relative to the control.</p><p>Integrated areas and the percent changes of O-H and N-H stretching bands (3500–3040 cm⁻¹) of the rat erythrocytes FTIR spectra of control and irradiated groups at 0, 4, 8, and 12 days postirradiation.</p

Percentage areas of protein secondary structures and their band assignments of rat erythrocytes FTIR spectra of the control and irradiated groups at 0, 4, 8, and 12 days postirradiation.

<p>Parameters (mean ± SD) were obtained by curve fitting of FTIR spectra,</p><p>^a significant change to the control values (p < 0.05).</p><p>Percentage areas of protein secondary structures and their band assignments of rat erythrocytes FTIR spectra of the control and irradiated groups at 0, 4, 8, and 12 days postirradiation.</p

CH₃, CH₂ asymmetric and symmetric stretching, amides A and B (3500–2800 cm⁻¹) of rats' erythrocytes sub-bands of control and irradiated groups at 4 days postirradiation. Wavenumbers, height of peaks, FWHHs, and areas corresponding to the resultant curve fitted Gaussian peaks.

<p>Parameters are expressed as the mean ± SD.</p><p>CH₃, CH₂ asymmetric and symmetric stretching, amides A and B (3500–2800 cm⁻¹) of rats' erythrocytes sub-bands of control and irradiated groups at 4 days postirradiation. Wavenumbers, height of peaks, FWHHs, and areas corresponding to the resultant curve fitted Gaussian peaks.</p

Erythrocytes FTIR spectra.

<p>Average of FTIR spectra exhibit rats erythrocytes obtained from the control and irradiated groups at 0, 4, 8, and 12 days postirradiation.</p

Ratios characteristics of the biomarkers derived from rat erythrocytes FTIR spectra of control and irradiated group at 4 days postirradiation.

<p>Parameters are expressed as the mean ± SD.</p><p>Ratios characteristics of the biomarkers derived from rat erythrocytes FTIR spectra of control and irradiated group at 4 days postirradiation.</p

FTIR spectra second derivative of erythrocytes CH stretching bands.

<p>Second derivative of the average of FTIR rat erythrocytes spectra in the range 3020–2800 cm⁻¹ obtained from the control and irradiated groups at 0, 4, 8, and 12 days postirradiation. Major bands were ν = (CH), νs(CH₃), νs(CH₂), νas(CH₃), and νas(CH₂).</p

Area of ester sub-bands C = O associated with phospholipids.

<p>The total area of ester sub-bands C = O associated with phospholipids in FTIR rat erythrocytes spectra of the control and irradiated groups as a function of the postirradiation time.</p

FTIR spectra second derivative of erythrocytes amides A and B.

<p>Second derivative of the average of FTIR rat erythrocytes spectra in the range (3500–3040 cm⁻¹) obtained from the control and irradiated groups at 0, 4, 8, and 12 days postirradiation. Major bands were ν = (OH), νas(OH), νas(NH), νs(NH), νs(OH), and ν = (NH).</p